PROJECT SUMMARY/ABSTRACT The risk of sudden unexpected death in epilepsy (SUDEP) in patients with epilepsy is more than 20-fold higher than that of death in the general population. Clinical and animal studies show that seizure-induced respiratory arrest is the primary event leading to death. Increased serotonin (5-HT) levels in the brain reduce seizure- induced respiratory arrest in provoked seizure models. However, it is unclear whether enhancing 5-HT neuro- transmission exerts protective effects on seizure-induced sudden death in spontaneous seizure (epilepsy) models and which 5-HT circuitry is involved in this sudden death in both provoked and spontaneous seizure models. These gaps in knowledge have significantly hindered the therapeutics to prevent SUDEP in patients. The long-term goal is to foster effective prevention strategies against SUDEP using approaches targeted to specific SUDEP mechanisms. The overall objectives of this proposal are to (1) determine the efficacy of 5-HT- enhancing agents in suppressing seizure-induced sudden death and (2) elucidate the involved 5-HT circuitry mechanisms in animal models, especially in a widely-used mouse model of human Dravet syndrome (a type of epilepsy) that displays spontaneous seizures with a high rate of seizure-induced sudden death. The central hypothesis is that enhanced 5-HT signaling prevents seizure-induced sudden death, and that the 5-HT raphe- amygdala circuitry is involved in this sudden death in DBA/1 and Dravet mice. The rationale for this proposal is that a determination of preclinical therapeutic efficacy of 5-HT-enhancing agents and 5-HT neuronal circuitry mechanisms in seizure-induced sudden death is likely to offer a strong scientific framework by which new strategies against human SUDEP can be developed. The central hypothesis will be tested in the following two specific aims: 1) Determine the protective effects of enhancing 5-HT neurotransmission on seizure-induced sudden death in DBA/1 and Dravet mice; and 2) Elucidate how 5-HT circuitry from raphe nuclei to the amygda- la modifies seizure-induced sudden death in DBA/1 and Dravet mice. We will employ a combination of simul- taneous video EEG/ECG/plethysmography monitoring, electrophysiology, pharmacology and cell-type specific technologies (optogenetics and DREADDs) to perform the work in these aims. The proposed research is inno- vative because it defines a novel 5-HT circuitry mechanism of seizure-induced sudden death using optogenet- ics and DREADDs in animal models, especially in a human disease model, which could conceptually advance the knowledge on the pathophysiological mechanisms of SUDEP. The proposed work is significant because the expected outcomes will potentially foster targeted pharmacologic and neurostimulatory interventions of SUDEP to save lives of at-risk patients.